土木学会第65回年次学術講演会(平成22年9月)
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Removal performance and mechanism for trace phenolic endocrine disruptors by aquatic plants.
Andre Rodrigues dos Reis1 and Yutaka Sakakibara*1
1. Introduction
Various chemical compounds have been discharged into the environment along with industrial activities
(Sakurai et al. 2004). Those having hormonal activities are called endocrine disrupting compounds (EDCs). The
performance of phytoremediation has proven effective in the removal of nutrients and metals from aqueous systems.
However, little information is available regarding EDCs and their removal pathways in aquatic environments. We
focused on the degradation of EDCs by peroxidase and hydrogen peroxide in aquatic plants. The objective of this
study was to investigate the possible effect of plant peroxidase and hydrogen peroxide (H2O2) on the removal of
EDCs by aquatic plants.
2. Materials and methods
Continuous experiment was conducted using Ceratophyllum demersum, Limnobium laevigatum and Fontinalis
antipyretica. The plants were cultivated in identical glass vessels containing 10% Hoagland’s nutrient solution (pH
6.0), where light intensity was kept at 350 µmol photons/m2/s. These plants were acclimatized to laboratory
conditions for more than 20 days, and were used for experiments. These plants were selected because of their wide
distribution in aquatic environment. One vessel without plant was prepared and used as a reference. The feed
concentration of EDCs was set at 100 µg/L. In addition, cell wall-bound peroxidase activities and plant endogenous
H2O2 were measured and relations to removal rates of EDCs were investigated.
Aquatic plants tissue were collected into liquid nitrogen and stored at -20 ºC. The measurements of H2O2
concentration and peroxidase activities were made according to former studies (Uchida et al., 2002; Pandolfini and
Gabbrielli, 1992).
3. Results and discussion
Figure 1 shows time course changes of these compounds by different aquatic plants. EDCs were effectively
removed by every aquatic plant in continuous treatment. However, pentachlorophenol was not removed
significantly by aquatic plants during the period in this study (data not shown).
Nonylphenol
80
Influent
Blank
CD
FA
LL
60
40
20
0
120
100
40
100
Influent
80
Blank
CD
60
FA
LL
40
20
20
Concentration (µg/L)
Concentration (µg/L)
Concentration (µg/L)
100
80
60
0
0
1 3
7 13 20 27 34
Time (days)
2,4-Dichlorophenol
4-tert-Octylphenol
120
120
Concentration (µg/L)
Bisphenol A
120
1
3
7 13 20 27 34
Time (days)
Influent
Blank
CD
FA
LL
40
20
0
0
0
100
Influent 80
Blank
CD
60
FA
LL
0
1
3
7 13 20 27 34
Time (days)
0 1 3 7 13 20 27 34
Time (days)
Figure 1. Time course changes of EDCs in continuous treatment.
To confirm the removal mechanism of EDCs, fresh biomass of aquatic plants was analyzed. After 34 days of
1
Department of Civil and Environmental Engineering. Waseda University. Shinjuku Ku, 3-4-1 Okubo, Tokyo 169 8555, Japan.
*Corresponding author. Tel/Fax: + 03-5286-3902. E-mail address: andrereis@aoni.waseda.jp
Keywords: Endocrine disrupting chemical, phytoremediation, environmental toxicology and aquatic plants.
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土木学会第65回年次学術講演会(平成22年9月)
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treatment, EDCs was scarcely detected in the aquatic plant (data not shown). This result indicates that aquatic
plants have no ability to absorb EDCs or accumulate it inside. These results suggest that these plants may possess
enzymes such as peroxidases that specifically metabolize EDCs containing a phenolic group. According to Figure 1,
the removal of EDCs did not occur in the absence of the plants (blank).
Figure 2 shows time course changes of plant peroxidase activity, demonstrating the enzyme activity differed
significantly among the plants. C. demersum had relatively larger soluble and ionically cell wall-bound peroxidase
activities (IPO). On the other hand, F. antipyretica increased the activities of IPO and covalently cell wall-bound
peroxidase (CPO) during the initial phase of experiment. Peroxidases are extremely versatile enzymes and best
known for their ability to utilize H2O2 in the oxidation of phenols.
Endogenous levels of H2O2 decreased with time. L. laevigatum and F. antipyretica showed relatively higher
levels of H2O2 (Figure 2). In the presence of toxic substance or under stressed conditions, plants may increase the
production of reactive oxygen species such as superoxide and H2O2, as a result of aerobic metabolism. It was
thought that H2O2 might be consumed by a complex enzymatic antioxidant system including catalase and
peroxidase.
C. demersum
L. laevigatum
F. antipyretica
SPO 180
IPO 160
CPO 140
120
100
80
60
40
20
0
200
150
100
50
0
0
5
10
20
Time (days)
30
0
5
10
20
700
H2O 2 (nmol/g FW)
△ ABS470nm/mg prot
250
12
10
8
6
4
600
500
400
300
200
2
100
0
0
30
0
5
10
20
Time (days)
Time (days)
CD
FA
LL
800
14
△ ABS470nm/mg prot
△ ABS470nm/mg prot
300
30
0
5
10
20
30
Time (days)
Figure 2. Time course changes of peroxidase activity and hydrogen peroxide, where: Soluble peroxidase activity
(SPO); Ionically cell wall-bound peroxidase activity (IPO); Covalently cell wall-bound peroxidase activity (CPO),
Ceratophyllum demersum (CD), Limnobium laevigatum (LL) and Fontinalis antipyretica (FA), respectively.
4. Conclusion
Experimental results demonstrated EDCs were effectively removed by different types of aquatic plants, except
pentachlorophenol. In addition, it was thought the removal rate might be affected by endogenous H2O2
concentration and peroxidase activity. Further studies will be need to analyze the ability of aquatic plants to remove
EDCs under different loading conditions.
Acknowledgment
This study was supported in part by a Research Found of JST Project on “Development for water reuse technology
in tropical region”.
References
Pandolfini T., Gabrielli R. (1992). Nickel toxicity and peroxidase activity in seedling of Triticum aestrivum L. Plant
Cell Environmental 15, 719-725.
Sakurai A., Toyoda S., Masuda M., Sakakibara M. (2004). Removal of bisphenol A by peroxidase-catalyzed
reaction using culture broth of Coprinus cinereus. Journal of Chemical Engineering of Japan, 37(2), 137-142.
Uchida A., Jagendorf A.T., Hibino T., Takabe T. (2002). Effects of hydrogen peroxide and nitric oxide on both salt
and heat stress tolerance in rice. Plant Science 163, 515-523.
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